Online Supplementary information for:

A modular RNA interference system for multiplexed gene regulation

Ari Dwijayanti1,2, Marko Storch1,2,3, Guy-Bart Stan2,4*, Geoff S. Baldwin1,2*

1 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK 2 Imperial College Centre for Synthetic Biology, Imperial College London, London SW7 2AZ, UK 3 London Biofoundry, Imperial College Translation & Innovation Hub, London W12 0BZ, UK 4 Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom

* To whom correspondence should be addressed. Email: [email protected]; [email protected]

Supplementary Fig. 1. Predicted base-pairing interactions of the designed mARi and mRNA target. Supplementary Fig. 2. Characterisation of constitutive promoters and transcript stoichiometry. Supplementary Fig. 3. Growth profile of different host strains expressing the mARi-based repression system. Supplementary Fig. 4. mARi-based regulation in different E. coli strains. Supplementary Fig. 5. Orthogonal repression by mARi. Supplementary Fig. 6. Multiplexed and simultaneous gene regulation by mARi. Supplementary Fig. 7. Maps for the plasmids used in this study.

Supplementary Table 1. Summary of mARi variants used to evaluate the impact of the position of the target site. Supplementary Table 2. UTR-RBS BASIC linker sequences used for testing the performance of mARi-A when combined with different RBS. Supplementary Table 3. The calculated relative expression ratio and repression activity of mARi. Supplementary Table 4. Orthogonal mARis and off-target prediction towards E. coli genome. Supplementary Table 5. List of standardised bioparts sequences used in this study. Supplementary Table 6. List of orthogonal BASIC linker sequences used in this study. UTR -A -RBSc a +1 5’ RiboJ RBS ATG..(sfGFP) 3’ Position 1 +82 +115 Position 2 +102 +127 Position 3 +111 +135 Position 4 +133 +155 b

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Supplementary Fig. 1. Predicted base-pairing interactions of the designed mARi and mRNA target.

(a) Schematic design of target site selection (Positions 1-4) of mARi-mediated repression. Arrows show the direction of the reverse complementary sequence in the mARi design. (b) Predicted base pairing of mARi-A with mRNA targets (sfGFP) for the four different target positions. Numbers indicate the relative positions of bases from the Transcription Start Site (+1). a b Promoter mARi Promoter mRNA

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Supplementary Fig. 2. Characterisation of constitutive promoters and transcript stoichiometry.

(a) Schematic of the expression system used to characterise our standardised constitutive promoter set. (b) Promoter activity for the set of the standardised constitutive promoters used for driving mARi and mRNA expression. This promoter activity was used to calculate the relative expression ratio (mARi:mRNA) in Fig. 3a. The subsets of promoters used for mRNA and mARi are indicated by the coloured wedges. (c) Normalised fluorescence from flow cytometry assay for all constructs used to evaluate transcript stoichiometry in Fig. 3c. Data are shown as the mean ± SD of three biological repeats (black dots). The calculated relative expression ratio and repression activity of mARi are provided in Supplementary Table 3.

Supplementary Fig. 3. Growth profile of different host strains expressing the mARi-based repression system.

Growth curves of strains with and without mARi expression in different host strains from a plate reader assay: (a) DH5α (K-12 strain), (b) DH10b (K-12 strain), (c) BL21(DE3) (B strain) and (d) BL21star(DE3) (B strain). Data associated with Fig. 3g were taken during early stationary phase (blue dotted line); Data associated with Fig. 3h were taken during mid-exponential phase at around 4h (orange dotted line) and early stationary phase at around 8h (blue dotted line). Lines show the mean from 3 independent measurements with the shaded area showing ± SD. Statistically significant differences were determined using Student’s t-test (ns used to denote “not significant”).

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Supplementary Fig. 4. mARi-based regulation in different E. coli strains.

The performance of mARi repression with a high expression ratio (mARi>mRNA), single plasmid system in four host strains was measured using a plate reader assay. Strains DH5α, DH10b, BL21(DE3), and BL21star(DE3) were used, with three different plasmid copy numbers: pSC101, p15A, and pMB1. Data were taken during early stationary phase (8 h). Data associated with Fig. 3g, h were taken from the p15A backbone. Data are shown as the mean ± SD of three independent repeats (black dots). a Target sequence b UTR -A UTR -B UTR -C UTR -D UTR -E 100

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Supplementary Fig. 5. Orthogonal repression by mARi.

(a) The calculated identity similarities of each target sequence in UTR pairs was obtained using the EMBOSS needle method (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) (b) Schematic of the genetic constructs used to evaluate target specificity of modular mARi-X and UTR-X pairs. The mRNA expression cassettes driven by PJ23101_BASIC were located in the p15A backbone while the mARi expression cassettes with PJ23119 were cloned in a pMB1 backbone. Both expression plasmids were then co-transformed into DH5α cells. (c) Response of all mARi and target sequence combinations were measured by flow cytometry assay after 6 h of incubation, and the fluorescence was normalised to control cells with sfGFP and without mARi. Data are shown as the mean ± SD of three independent repeats (black dots). Statistically significant differences were determined using Student’s t-test (**** represents p<0.0001, ** represents p<0.01).

Supplementary Fig. 6. Multiplexed and simultaneous gene regulation by mARi.

Measuring the response of mARi regulation in multi-gene systems: independent transcriptional units of two-gene (a) and three-gene (b), and of operon constructs (c). In each case sfGFP was downstream of UTR-A-RBSc, mCherry was downstream of UTR-B-RBS-c, and mTagBFP was downstream UTR-C- RBSc. mARi-A, mARi-B, and mARi-C were expressed both separately and together and their effect on fluorescent protein output was measured by flow cytometry assay. Data are shown as the mean ± SD of three biological repeats (black dots); flow cytometry histograms are also shown.

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(a) Map of a plasmid containing a single transcriptional unit of the UTR-A-RBSc-sfGFP expression cassette. (b) Map of a plasmid harbouring the PJ23101 mARi repression system that was used for identifying target site position, transcript stoichiometry, and spatial organisation. (c) Map of a single mARi expression plasmid. (d) Map of a double mARi expression plasmid. (e) Map of a triple mARi expression plasmid. (f) Map of a plasmid containing dual transcriptional units for UTR-A-RBSc-sfGFP and UTR-B-RBSc-mCherry. (g) Map of a plasmid containing triple transcriptional units for UTR-A-RBSc- sfGFP, UTR-B-RBSc-mCherry, and UTR-C-RBSc-mTagBFP. (h) Map of a plasmid containing an operon system with UTR-A-RBSc sfGFP and UTR-B-RBSc mCherry. (i) Map of an empty plasmid containing an origin of replication and antibiotic resistance genes. Details of BASIC bioparts used to build the plasmids are provided in Supplementary Table 5. The list of orthogonal linkers used to construct the plasmids using BASIC is provided in Supplementary Table 6.

Supplementary Table 1. Summary of mARi variants used to evaluate the impact of the position of the target site.

Parameter mARi-A targeting mARi-A targeting mARi-A targeting mARi-A targeting Position 1 Position 2 Position 3 Position 4 Context dependencya Specific to the Yes* Yes* Yes* No upstream region of UTR sequence Specific to the No* Yes Yes No* RBS sequence Specific to the No* No* No* Yes GOI sequence Target 5’- 5’- 5’- 5’- sequenceb GTTGAACACCGTCT GTATCAGTTGTAA GTAAAAAGAGGG ATGCGTAAAGGCG CAGGTAAGTATCAG AAAGAGGGGAAA GAAATAGTCCAT AAGAACTGTT-3’ TTGTAAA-3’ T-3’ G-3’ GC content 40 34.62 40 43.8 (%) Length (bp) 35 26 25 23 Free binding -55.2 -36.8 -36.7 -35.7 energy (kcal/mol)c mARi TTTACAACTGATAC ATTTCCCCTCTTTT CATGGACTATTTC AACAGTTCTTCGC sequenced TTACCTGAGACGGT TACAACTGATACttt CCCTCTTTTTACttt CTTTACGCATtttct GTTCAACtttctgttgg ctgttgggccattgcatt ctgttgggccattgcat gttgggccattgcattgc gccattgcattgccactga gccactgattttccaaca tgccactgattttccaa cactgattttccaacata ttttccaacatataaaaa tataaaaagacaagcc catataaaaagacaa taaaaagacaagcccg gacaagcccgaacagtc cgaacagtcgtccgggc gcccgaacagtcgtcc aacagtcgtccgggctt gtccgggcttttttt ttttttt gggcttttttt ttttt a The dependency of the target site position towards different contexts is highlighted. * denotes highly desirable for modularity. b Target sequence within the UTR-RBS BASIC linker (1) used in a sfGFP expression cassette. c The free binding energy of the base-pairing region was calculated at 37˚C (see Methods). d Seed sequences are shown in uppercase, while the MicC sRNA scaffold is shown in lowercase.

Supplementary Table 2. UTR-RBS BASIC linker sequences used for testing the performance of mARi-A when combined with different RBS.

UTR-RBS Sequencea Mean relative Predicted strength linker expression RBS calculator EMOPEC v2.0 (2, 3) (4) UTR-A-RBSa ggctcgttgaacaccgtctcaggtaagtatc 1.00 715277.31 77.8% agttgtaaaaagaggagaaatagtcc UTR-A-RBSb ggctcgttgaacaccgtctcaggtaagtatc 0.52 673992.76 80.5% agttgtaaatctaaggaggtagtcc UTR-A-RBSc ggctcgttgaacaccgtctcaggtaagtatc 0.35 689020.88 79.5% agttgtaaaaagaggggaaatagtcc UTR-A-RBSd ggctcgttgaacaccgtctcaggtaagtatc 0.23 17315.58 90.5% agttgtaaatcccaggaggtagtcc UTR-A-RBSe ggctcgttgaacaccgtctcaggtaagtatc 0.18 715277.31 77.3% agttgtaaatctcgggaggtagtcc Predicted output values are shown for computational evaluation of the “strength” of UTR-RBS sequences including the first 100 bases of the sfGFP reporter DNA sequence using two different methods (RBS calculator v2.0 and EMOPEC). a DNA sequence colours correspond to upstream scar (blue) and downstream scar (orange). The DNA sequence in underlined-bold indicates the RBS sequence.

Supplementary Table 3. The calculated relative expression ratio and repression activity of mARi.

mARi mRNA Calculated relative expression Experimentally verified promoter promoter ratio (mARi/mRNA) normalized fluorescence J23119 J23111 0.424654 0.444787 ± 0.004998 J23111 J23111 0.237425 0.461128 ± 0.023085 J23104 J23111 0.150723 0.493309 ± 0.015242 J23101 J23111 0.000199 0.615604 ± 0.041495 J23119 J23104 0.561215 0.306795 ± 0.024429 J23111 J23104 0.313776 0.344862 ± 0.031806 J23104 J23104 0.199192 0.393503 ± 0.017245 J23101 J23104 0.000263 0.521379 ± 0.029825 J23119 J23101 1.410095 0.222778 ± 0.010729 J23111 J23101 0.788385 0.275617 ± 0.016233 J23104 J23101 0.500486 0.292916 ± 0.018146 J23101 J23101 0.000662 0.378971 ± 0.041516 J23119 J23108 4.48818 0.274005 ± 0.025352 J23111 J23108 2.509346 0.29306 ± 0.033318 J23104 J23108 1.592994 0.291721 ± 0.013977 J23101 J23108 0.002107 0.358764 ± 0.00956 Supplementary Table 4. Orthogonal mARis and off-target prediction towards E. coli genome.

mARi Seed GC Free binding mARi sequencec Off-target predictiond sequencea content of energy of (35bp) seed seed Rank Free binding Gene annotation sequence sequenceb energy of base- (kcal/mol) pairing interaction (kcal/mol) mARi-A TTTACAACTGA 40 % -55.2 TTTACAACTGATACTTACCTG 1 -27.95 RNA pyrophosphohydrolase TACTTACCTGA AGACGGTGTTCAACtttctgttg GACGGTGTTCA ggccattgcattgccactgattttcca 2 -28.08 YhcH/YigK/YiaL family protein AC acatataaaaagacaagcccgaaca 3 -25.69 16S rRNA (cytosine(1407)-C(5))- gtcgtccgggcttttttt methyltransferase RsmF mARi-B TTACAATAGAT 40 % -54.9 TTACAATAGATTTTACCGTCA 1 -33.85 DUF386 family protein cupin superfamily TTTACCGTCAG GACCACGAGATACCtttctgttg 2 -25.10 16S rRNA m(5)C1407 methyltransferase ACCACGAGATA ggccattgcattgccactgattttcca SAM-dependent CC acatataaaaagacaagcccgaaca 3 -19.07 DUF554 family putative inner membrane gtcgtccgggcttttttt protein mARi-C TTTTCTGCTACC 40 % -55.8 TTTTCTGCTACCCTTATCTCA 1 -27.42 NADH:ubiquinone oxidoreductase CTTATCTCAGC GCCAATAGTAACACtttctgttg membrane subunit J CAATAGTAACA ggccattgcattgccactgattttcca 2 -27.04 Sensory histidine kinase in two-component C acatataaaaagacaagcccgaaca system with NarP gtcgtccgggcttttttt 3 -27.03 DUF386 family protein cupin superfamily mARi-D TTTACATAGAA 42.86 % -57.2 TTTACATAGAATACACAGCC 1 -27.06 16S rRNA m(5)C1407 methyltransferase TACACAGCCGG GGGACAGGGTATAACtttctgt SAM-dependent GACAGGGTATA tgggccattgcattgccactgattttcc 2 -26.28 DUF386 family protein cupin superfamily AC aacatataaaaagacaagcccgaac 3 -23.62 O-antigen capsule forming protein-tyrosine- agtcgtccgggcttttttt phosphatase;Etk-P dephosphorylase mARi-E TTTACATATGTT 31.43 % -48.9 TTTACATATGTTTTATCGTCA 1 -28.14 DUF386 family protein cupin superfamily TTATCGTCAAG AGACGCTGTATAACtttctgttg 2 -23.97 Enterobactin/ferrienterobactin esterase ACGCTGTATAA ggccattgcattgccactgattttcca 3 -21.20 16S rRNA m(5)C1407 methyltransferase C acatataaaaagacaagcccgaaca SAM-dependent gtcgtccgggcttttttt a Seed sequence in the mARi cassette is the reverse complementary sequence for the cognate target sites within the UTR-RBS BASIC linker (1). b The free binding energy of the base-pairing region was calculated at 37˚C (see Methods). c Seed sequences are shown in uppercase, while the MicC sRNA scaffold is shown in lowercase. d The off-target effect of full sequence of mARis was computed using CopraRNA (5–7) against NC_000913 (E. coli MG1655), NC_010473 (E. coli DH10b), NC_012892 and NC_012971 (E. coli BL21(DE3)) as host references (see Methods).

Supplementary Table 5. List of standardised bioparts sequences used in this study.

Part name Type Sequencea (5’3’) Genbank accession number

PJ23119_BASIC Standardised tctggtgggtctctgtccCCAATTATTGAACACCCTTCGGGGTGTTTTTTTGTTTCTGGTCTA constitutive CCATCTCGTTGTGATAATAGACCTGAAGTGCCTACTCTGGAAAATCTTTGACAGCTA promoterb GCTCAGTCCTAGGTATAATGCTAGCAGCTGTCACCGGATGTGCTTTCCGGTCTGATG AGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgggagacctatcg PJ23111_BASIC Standardised tctggtgggtctctgtccCCAATTATTGAACACCCTTCGGGGTGTTTTTTTGTTTCTGGTCTA constitutive CCATCTCGTTGTGATAATAGACCTGAAGTGCCTACTCTGGAAAATCTTTGACGGCTA promoterb GCTCAGTCCTAGGTATAGTGCTAGCAGCTGTCACCGGATGTGCTTTCCGGTCTGATG AGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgggagacctatcg PJ23104_BASIC Standardised tctggtgggtctctgtccCCAATTATTGAACACCCTTCGGGGTGTTTTTTTGTTTCTGGTCTA constitutive CCATCTCGTTGTGATAATAGACCTGAAGTGCCTACTCTGGAAAATCTTTGACAGCTA promoterb GCTCAGTCCTAGGTATTGTGCTAGCAGCTGTCACCGGATGTGCTTTCCGGTCTGATG AGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgggagacctatcg PJ23101_BASIC Standardised tctggtgggtctctgtccCCAATTATTGAACACCCTTCGGGGTGTTTTTTTGTTTCTGGTCTA constitutive CCATCTCGTTGTGATAATAGACCTGAAGTGCCTACTCTGGAAAATCTTTTACAGCTAG promoterb CTCAGTCCTAGGTATTATGCTAGCAGCTGTCACCGGATGTGCTTTCCGGTCTGATGA GTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgggagacctatcg PJ23108_BASIC Standardised tctggtgggtctctgtccCCAATTATTGAACACCCTTCGGGGTGTTTTTTTGTTTCTGGTCTA constitutive CCATCTCGTTGTGATAATAGACCTGAAGTGCCTACTCTGGAAAATCTCTGACAGCTA promoterb GCTCAGTCCTAGGTATAATGCTAGCAGCTGTCACCGGATGTGCTTTCCGGTCTGATG AGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAggctcgggagacctatcg PJ23101_mARi-A Position 1 mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT (J23101_mARi-A) cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAGTTGAACACCGTCTCAGGTAAGTATCAGTTGTAAAGCTAGCATAATACCTA GGACTGAGCTAGCTGTAAAggctcgggagacctatcg PJ23101_mARi-A Position 2 mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAGTATCAGTTGTAAAAAGAGGGGAAATGCTAGCATAATACCTAGGACTGAG CTAGCTGTAAAggctcgggagacctatcg PJ23101_mARi-A Position 3 mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAGTAAAAAGAGGGGAAATAGTCCATGGCTAGCATAATACCTAGGACTGAG CTAGCTGTAAAggctcgggagacctatcg PJ23101_mARi-A Position 4 mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAATGCGTAAAGGCGAAGAACTGTTGCTAGCATAATACCTAGGACTGAGCTA GCTGTAAAggctcgggagacctatcg PJ23119_mARi-A mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAGTTGAACACCGTCTCAGGTAAGTATCAGTTGTAAAGCTAGCATTATACCTA GGACTGAGCTAGCTGTCAAggctcgggagacctatcg PJ23111_mARi-A mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAGTTGAACACCGTCTCAGGTAAGTATCAGTTGTAAAGCTAGCACTATACCTA GGACTGAGCTAGCCGTCAAggctcgggagacctatcg PJ23104_mARi-A mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAGTTGAACACCGTCTCAGGTAAGTATCAGTTGTAAAGCTAGCACAATACCTA GGACTGAGCTAGCTGTCAAggctcgggagacctatcg PJ23119_mARi-B mARi expression tctggtgggtctctgtccGGACCAAAACGAAAAAACACCCTTTCGGGTGTCTTTTCTGGAAT cassette TTGGTACCGAGAAAAAAAGCCCGGACGACTGTTCGGGCTTGTCTTTTTATATGTTGG AAAATCAGTGGCAATGCAATGGCCCAACAGAAAGGTATCTCGTGGTCTGACGGTAA AATCTATTGTAAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAggctcgggagacc tatcg PJ23119_mARi-C mARi expression tctggtgggtctctgtccTATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACT cassette CTAGTAGAGAGCGTTCACCGACAAAAACAGATAAAACGAAAGGCCCAGTCTTTCGA CTGAGCCTTTCGTTTTATTTGATGCCTGGCTCGAGCTCGAGAAAAAAAGCCCGGACG ACTGTTCGGGCTTGTCTTTTTATATGTTGGAAAATCAGTGGCAATGCAATGGCCCAA CAGAAAGTGTTACTATTGGCTGAGATAAGGGTAGCAGAAAAGCTAGCATTATACCT AGGACTGAGCTAGCTGTCAAggctcgggagacctatcg PJ23119_mARi-D mARi expression tctggtgggtctctgtccGGACCAAAACGAAAAAACACCCTTTCGGGTGTCTTTTCTGGAAT cassette TTGGTACCGAGAAAAAAAGCCCGGACGACTGTTCGGGCTTGTCTTTTTATATGTTGG AAAATCAGTGGCAATGCAATGGCCCAACAGAAAGTTATACCCTGTCCCGGCTGTGT ATTCTATGTAAAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAGATTTTCCA GAGTAGGCACTTCAGGTCTATTATCACAACGAGATGGTAGACCAGAAACAAAAAAA CACCCCGAAGGGTGTTCAATAATTGGggctcgggagacctatcg PJ23119_mARi-E mARi expression tctggtgggtctctgtccGGACCAAAACGAAAAAACACCCTTTCGGGTGTCTTTTCTGGAAT cassette TTGGTACCGAGAAAAAAAGCCCGGACGACTGTTCGGGCTTGTCTTTTTATATGTTGG AAAATCAGTGGCAATGCAATGGCCCAACAGAAAGTTATACAGCGTCTTGACGATAA AACATATGTAAAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAGATTTTCCA GAGTAGGCACTTCAGGTCTATTATCACAACGAGATGGTAGACCAGAAACAAAAAAA CACCCCGAAGGGTGTTCAATAATTGGggctcgggagacctatcg sfGFP_no terminator Reporter tctggtgggtctctgtccATGCGTAAAGGCGAAGAACTGTTCACGGGCGTAGTTCCGATTC TGGTCGAGCTGGACGGCGATGTGAACGGTCATAAGTTTAGCGTTCGCGGTGAAGGT GAGGGCGACGCGACCAACGGCAAACTGACCCTGAAGTTCATCTGCACCACCGGTAA ACTGCCGGTGCCTTGGCCGACCTTGGTGACGACGTTGACGTATGGCGTGCAGTGTT TTGCGCGTTATCCGGACCACATGAAACAACACGATTTCTTCAAATCTGCGATGCCGG AGGGTTACGTCCAGGAGCGTACCATTTCCTTCAAGGATGATGGCACTTACAAAACTC GCGCAGAGGTTAAGTTTGAAGGTGACACGCTGGTCAATCGTATCGAATTGAAGGGT ATCGACTTTAAAGAGGATGGTAACATTCTGGGCCATAAACTGGAGTATAACTTCAAC AGCCATAATGTTTACATTACGGCAGACAAGCAAAAGAACGGCATCAAGGCCAATTT CAAGATTCGCCACAATGTTGAGGACGGTAGCGTCCAACTGGCCGACCATTACCAGC AGAACACCCCAATTGGTGACGGTCCGGTTTTGCTGCCGGATAATCACTATCTGAGCA CCCAAAGCGTGCTGAGCAAAGATCCGAACGAAAAACGTGATCACATGGTCCTGCTG GAATTTGTGACCGCTGCGGGCATCACCCACGGTATGGACGAGCTGTATAAGCGTCC GTAAggctcgggagacctatcg sfGFP_B15 Reporter tctggtgggtctctgtccATGCGTAAAGGCGAAGAACTGTTCACGGGCGTAGTTCCGATTC TGGTCGAGCTGGACGGCGATGTGAACGGTCATAAGTTTAGCGTTCGCGGTGAAGGT GAGGGCGACGCGACCAACGGCAAACTGACCCTGAAGTTCATCTGCACCACCGGTAA ACTGCCGGTGCCTTGGCCGACCTTGGTGACGACGTTGACGTATGGCGTGCAGTGTT TTGCGCGTTATCCGGACCACATGAAACAACACGATTTCTTCAAATCTGCGATGCCGG AGGGTTACGTCCAGGAGCGTACCATTTCCTTCAAGGATGATGGCACTTACAAAACTC GCGCAGAGGTTAAGTTTGAAGGTGACACGCTGGTCAATCGTATCGAATTGAAGGGT ATCGACTTTAAAGAGGATGGTAACATTCTGGGCCATAAACTGGAGTATAACTTCAAC AGCCATAATGTTTACATTACGGCAGACAAGCAAAAGAACGGCATCAAGGCCAATTT CAAGATTCGCCACAATGTTGAGGACGGTAGCGTCCAACTGGCCGACCATTACCAGC AGAACACCCCAATTGGTGACGGTCCGGTTTTGCTGCCGGATAATCACTATCTGAGCA CCCAAAGCGTGCTGAGCAAAGATCCGAACGAAAAACGTGATCACATGGTCCTGCTG GAATTTGTGACCGCTGCGGGCATCACCCACGGTATGGACGAGCTGTATAAGCGTCC GTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGG GCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCT CACCTTCGGGTGGGCCTTTCTGCGTTTATAggctcgggagacctatcg sfGFP-ECK120033737_Term Reporter tctggtgggtctctgtccATGCGTAAAGGCGAAGAACTGTTCACGGGCGTAGTTCCGATTC TGGTCGAGCTGGACGGCGATGTGAACGGTCATAAGTTTAGCGTTCGCGGTGAAGGT GAGGGCGACGCGACCAACGGCAAACTGACCCTGAAGTTCATCTGCACCACCGGTAA ACTGCCGGTGCCTTGGCCGACCTTGGTGACGACGTTGACGTATGGCGTGCAGTGTT TTGCGCGTTATCCGGACCACATGAAACAACACGATTTCTTCAAATCTGCGATGCCGG AGGGTTACGTCCAGGAGCGTACCATTTCCTTCAAGGATGATGGCACTTACAAAACTC GCGCAGAGGTTAAGTTTGAAGGTGACACGCTGGTCAATCGTATCGAATTGAAGGGT ATCGACTTTAAAGAGGATGGTAACATTCTGGGCCATAAACTGGAGTATAACTTCAAC AGCCATAATGTTTACATTACGGCAGACAAGCAAAAGAACGGCATCAAGGCCAATTT CAAGATTCGCCACAATGTTGAGGACGGTAGCGTCCAACTGGCCGACCATTACCAGC AGAACACCCCAATTGGTGACGGTCCGGTTTTGCTGCCGGATAATCACTATCTGAGCA CCCAAAGCGTGCTGAGCAAAGATCCGAACGAAAAACGTGATCACATGGTCCTGCTG GAATTTGTGACCGCTGCGGGCATCACCCACGGTATGGACGAGCTGTATAAGCGTCC GTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGGGAAACACAGAAAAAA GCCCGCACCTGACAGTGCGGGCTTTTTTTTTCGACCAAAGGTACTggctcgggagacctat cg mCherry-B15_Term Reporter tctggtgggtctctgtccATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAG GAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGA GATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTG AAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTC ATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAG CTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGG CGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGG TGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACC ATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGG GCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGT CAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCA ACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACG AACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATA ATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTT CGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTT CGGGTGGGCCTTTCTGCGTTTATAggctcgggagacctatcg mCherry-B14_Term Reporter tctggtgggtctctgtccATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAG GAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGA GATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTG AAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTC ATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAG CTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGG CGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGG TGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACC ATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGG GCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGT CAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCA ACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACG AACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAAGG CTCGATCACGGCACTACACTCGTTGCTTTATCGGTATTGTTATTACAGAGTCCTCACA CTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGAGAGAATATAAAA AGCCAGATTATTAATCCGGCTTTTTTATTATTTggctcgggagacctatcg mTagBFP-B15_term Reporter tctggtgggtctctgtccATGTCCGAGTTGATCAAAGAGAACATGCATATGAAATTATATAT GGAAGGCACTGTAGATAATCATCATTTTAAATGTACGTCGGAAGGCGAAGGTAAAC CATATGAAGGTACGCAGACGATGCGCATCAAGGTGGTGGAGGGCGGTCCGCTGCC ATTCGCTTTCGATATTTTAGCCACGAGCTTCCTCTACGGTTCTAAAACTTTCATCAATC ACACGCAGGGTATTCCGGACTTCTTTAAACAGTCGTTCCCGGAGGGTTTCACCTGGG AACGCGTTACCACGTATGAAGATGGTGGTGTGCTTACGGCAACGCAGGACACGAGC CTTCAGGATGGGTGTTTGATTTACAACGTGAAAATTCGTGGTGTGAACTTCACGTCT AACGGCCCGGTGATGCAGAAAAAAACACTGGGTTGGGAAGCCTTTACCGAAACCCT GTATCCGGCGGACGGTGGCCTGGAAGGCCGTAATGATATGGCCTTGAAATTAGTCG GCGGTTCACACCTGATCGCGAACGCGAAAACAACCTATCGTAGTAAAAAACCAGCC AAAAACCTGAAAATGCCGggcGTCTACTACGTAGACTACCGTCTGGAGCGCattAAAG AGGCGAATAATGAAACCTATGTCGAGCAGCACGAAGTTGCGGTTGCACGCTATTGC GATCTGCCCAGCAAACTGGGCCACAAGCTTAATGGTAGCTAATAATACTAGAGCCA GGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTT GTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTT TCTGCGTTTATAggctcgggagacctatcg a Coloured, lowercase DNA sequences correspond to prefix (blue) and suffix (orange). Bold DNA sequences indicate the start codon. b Standardised constitutive promoters consist of a synthetic terminator (L3S3P11) (8, 9), up element, core Anderson constitutive promoter (parts.igem.org), and ribozyme insulator (RiboJ) (10). The terminator L3S3P11 used in the standardised promoter was mutated to remove the BsaI site (9).

Supplementary Table 6. List of orthogonal BASIC linker sequences used in this study.

BASIC linker Type Sequencea (5’3’) UTR-A-RBSa UTR-RBS linker ggctcgttgaacaccgtctcaggtaagtatcagttgtaaaaagaggagaaatagtcc UTR-A-RBSb UTR-RBS linker ggctcgttgaacaccgtctcaggtaagtatcagttgtaaatctaaggaggtagtcc UTR-A-RBSc UTR-RBS linker ggctcgttgaacaccgtctcaggtaagtatcagttgtaaaaagaggggaaatagtcc UTR-A-RBSd UTR-RBS linker ggctcgttgaacaccgtctcaggtaagtatcagttgtaaatcccaggaggtagtcc UTR-A-RBSe UTR-RBS linker ggctcgttgaacaccgtctcaggtaagtatcagttgtaaatctcgggaggtagtcc UTR-B-RBSc UTR-RBS linker ggctcggtatctcgtggtctgacggtaaaatctattgtaaaagaggggaaatagtcc UTR C-RBSc UTR-RBS linker ggctcgtgttactattggctgagataagggtagcagaaaaaagaggggaaatagtcc UTR D-RBSc UTR-RBS linker ggctcgttataccctgtcccggctgtgtattctatgtaaaaagaggggaaatagtcc UTR E-RBSc UTR-RBS linker ggctcgttatacagcgtcttgacgataaaacatatgtaaaaagaggggaaatagtcc MLA Methylated linker ggctcgggtaagaactcgcacttcgtggaaacactattatctggtgggtctctgtcc MLB Methylated linker ggctcgggagacctatcggtaataacagtccaatctggtgtaacttcggaatcgtcc L1 Neutral linker ggctcgttacttacgacactccgagacagtcagagggtatttattgaactagtcc L2 Neutral linker ggctcgatcggtgtgaaaagtcagtatccagtcgtgtagttcttattacctgtcc L3 Neutral linker ggctcgatcacggcactacactcgttgctttatcggtattgttattacagagtcc L4 Neutral linker ggctcgacccacgactattgactgctctgagaaagttgattgttacgattagtcc L5 Neutral linker ggctcgagaagtagtgccacagacagtattgcttacgagttgatttatcctgtcc a DNA sequence colours correspond to upstream scar (blue) and downstream scar (orange). The DNA sequence in underlined-bold indicates the RBS sequence.

Supplementary references

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